Theme: Microbiological diagnosis of anaerobic diseases.

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Theme: Microbiological diagnosis of anaerobic diseases

Spore- forming (Clostridia) Nonspore-forming Gram-negativeGram-positive C.botulinumRod-shaped C.tetaniBacteroidesActinomyces C.perfringensPrevotellaBifidobacterium C.novyiFusobacteriumEubacterium C.septicumLeptotrichiaPropionibacterium C.histolyticumCocci C.dificilleVeilonellaPeptococcus (about 100 species) Peptostreptococcus Spiral forms Treponema, Borrelia

Effects of oxygen on aerobic, anaerobic, and facultative anaerobic bacteria

Chemical dynamics in anaerobic bacteria

Anaerobic culture system (anaerobic jar or GasPak jar)

Nutrient media Milk under oilKitt-Tarozzi medium Wilson-Blair agar Sugar blood agar

Family Bacillaceae Genus Clostridium Causative agents: tetanus - C.tetani gas gangrene: C.perfringens, C.novyi, C.septicum, C.histolyticum botulism - C.botulinum pseudomembranous enterocolitis -C.difficile

C.tetani. Tetanus risus sardonicus opisthotonos Trismus or lockjaw

Mechanism of Action of Tetanus Toxin

Clostridia of gas gangrene C.histolyticum С. septicum C.novyiC.perfringens

Micro & Macroscopic C. perfringens NOTE: Large rectangular gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin NOTE: Double zone of hemolysis

C. perfringens Virulence Factors Major Minor

C. perfringens Nagler Reaction NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α-toxin inhibits activity around left streak.

Gas gangrene

Microbiological diagnosis of anaerobic infection Specimen: wound exudate, edema liquid, necrotic tissues. Methods: 1.IF. 2.Microscopy. 3.Bacteriological. 4.Biological (neutralization test). 5.PCR.

Scheme of microbiological diagnosis of anaerobic infection 1 step 2 step 3 step 4 step Microscopic examination Bacteriological method Biological method Gram stain result Express test – lecitinase test Seeding in Kitt- Tarozzi medium, milk under oil result Gram stain Seeding on sugar- blood agar in Petri dish Guinea pig inoculation Morphology of colonies, Gram stain Seeding in Kitt-Tarozzi medium resultSeeding in Hiss media Neutralization test to detect exotoxin (mouse inoculation) Definitive diagnosis Specimen: wound exudate, edema liquid, necrotic tissues

Lecitinase test Lecitinase positive colonies of C.perfringens on egg yolk agar

Differentiation of Clostridia due to culture properties Sugar-blood agar: –С. perfringens –С. novyi –С. septicum –С. histolyticum

C. botulinum Gram stain Growth on sugar-blood agar

Botulism toxin

Mechanism of Action of Botulinum Toxin

Botulism toxin action

Botulism Ptosis Wound botulism

Laboratory diagnosis of botulism Specimens: food, feces, vomit, may be patients blood. Culture: Isolation of organism. Biological: Demonstration of C.botulinum toxin on the guinea pigs. Detection of toxin: PHAT.

Scheme of laboratory diagnosis of botulism Culture

Scheme of laboratory diagnosis of botulism

Neutralization test for detection of toxin Control (specimen) Specimen + antibodies against toxin А Specimen + antibodies against toxin В Specimen + antibodies against toxin E

BoNT A (Botox) Botox injection patient 13 weeks after injection Sadick, N. and A.R. Herman (2003). Comparison of Botulinum Toxins A and B in the Aesthetic Treatment of Facial Rhytides. Dermatologic Surgery 29:

C. difficile

C. dificile Virulence Factors

Laboratory diagnosis of pseudomembranous enterocolitis Specimens: feces. Culture: Isolation of organism. Detection of toxin: ELISA, cell culture. PCR.